Engineering of a Novel Saccharomyces cerevisiae Wine Strain with a Respiratory Phenotype at High External Glucose Concentrations

Abstract

The recently described respiratory strain Saccharomyces cerevisiae KOY.TM6*P is, to our knowledge, the only reported strain of S. cerevisiae which completely redirects the flux of glucose from ethanol fermentation to respiration, even at high external glucose concentrations (27). In the KOY.TM6*P strain, portions of the genes encoding the predominant hexose transporter proteins, Hxt1 and Hxt7, were fused within the regions encoding transmembrane (TM) domain 6. The resulting chimeric gene, TM6*, encoded a chimera composed of the amino-terminal half of Hxt1 and the carboxy-terminal half of Hxt7. It was subsequently integrated into the genome of an hxt null strain. In this study, we have demonstrated the transferability of this respiratory phenotype to the V5 hxt1-7⌬ strain, a derivative of a strain used in enology. We also show by using this mutant that it is not necessary to transform a complete hxt null strain with the TM6* construct to obtain a nonethanol-producing phenotype. The resulting V5.TM6*P strain, obtained by transformation of the V5 hxt1-7⌬ strain with the TM6* chimeric gene, produced only minor amounts of ethanol when cultured on external glucose concentrations as high as 5%. Despite the fact that glucose flux was reduced to 30% in the V5.TM6*P strain compared with that of its parental strain, the V5.TM6*P strain produced biomass at a specific rate as high as 85% that of the V5 wild-type strain. Even more relevant for the potential use of such a strain for the production of heterologous proteins and also of low-alcohol beverages is the observation that the biomass yield increased 50% with the mutant compared to its parental strain.

Elevated Ras/protein kinase A activity in Saccharomyces cerevisiae reduces proliferation rate and lifespan by two different reactive oxygen species‐dependent routes

“…It is not clear how, exactly, a reduction in ATP/ADP carrier function impedes the growth rate of cells. Under the glucoserepressive growth conditions used in this work, ATP is generated predominantly from substrate level phosphorylation (Larsson et al, 1998;Rolland et al, 2002;Henricsson et al, 2005). However, residual respiration is proceeding under such conditions and, taking respiratory rate and efficiency, that is, P/O ratios, into account, it has been calculated that about 30% of the ATP is derived from mitochondrial respiration in glucose-repressed cells (Larsson et al, 1998).…”

Reducing alcohol levels in wines through rational and evolutionary engineering of Saccharomyces cerevisiae

“…By contrast to the classical respirofermentative catabolism of S. cerevisiae strains, this strain shows a fully respiratory metabolism at high glucose levels. Thus, part of the glucose can be used for respiration, resulting in a decreased ethanol yield and increased biomass formation (Henricsson et al, 2005). However, this strain switches to fermentation when oxygen is lacking, which hinders its use during fermentative processes such as wine fermentation.…”

Control of alcoholic fermentation in winemaking: Current situation and prospect

“…The high level of ethanol in some wines has led to problems worldwide. Several strategies for reducing ethanol yield have recently been tested: glycerol overproduction (Cambon, Monteil, Remize, & Dequin, 2006), NADH oxidase expression (Heux, Sablayrolles, Cachon, & Dequin, 2006), use of a respiratory strain (Henricsson et al, 2005), glucose oxidase expression (Malherbe, du Toit, Cordero Otero, van Rensburg, & Pretorius, 2003).…”